The present invention relates generally to oligonucleotides and more specifically to oligonucleotides which have a sequence including at least one unmethylated CpG dinucleotide which are immunostimulatory.
In the 1970s, several investigators reported the binding of high molecular weight DNA to cell membranes (Lerner, R. A., et al. 1971. xe2x80x9cMembrane-associated DNA in the cytoplasm of diploid human lymphocytes.xe2x80x9d Proc. Natl. Acad. Sci. USA 68:1212; Agrawal, S. K., R. W. Wagner, P. K. McAllister, and B. Rosenberg. 1975. xe2x80x9cCell-surface-associated nucleic acid in tumorigenic cells made visible with platinum-pyrimidine complexes by electron microscopy.xe2x80x9d Proc. Natl. Acad. Sci. USA 72:928). In 1985, Bennett et al. presented the first evidence that DNA binding to lymphocytes is similar to a ligand receptor interaction: binding is saturable, competitive, and leads to DNA endocytosis and degradation into oligonucleotides (Bennett, R. M., G. T. Gabor, and M. M. Merritt, 1985. xe2x80x9cJ. Clin. Invest. 76:2182). Like DNA, oligodeoxyribonucleotides (ODNs) are able to enter cells in a saturable, sequence independent, and temperature and energy dependent fashion (reviewed in Jaroszewski, J. W., and J. S. Cohen. 1991. xe2x80x9cCellular uptake of antisense oligodeoxynucleotides.xe2x80x9d Advanced Drug Deliver Reviews 6:235; Akhtar, S., Y. Shoji, and R. L. Juliano. 1992. xe2x80x9cPharmaceutical aspects of the biological stability and membrane transport characteristics of antisense oligonucleotides.xe2x80x9d In: Gene Regulation: Biology of Antisense RNA and DNA. R. P. Erickson, and J. G. Izant, eds. Raven Press, Ltd. New York, pp. 133; and Zhao, Q., T. Waldschmidt, E. Fisher, C. J. Herrera, and A. M. Krieg. 1994. xe2x80x9cStage specific oligonucleotide uptake in murine bone marrow B cell precursors.xe2x80x9d Blood 84:3660). No receptor for DNA or ODN uptake has yet been cloned, and it is not yet clear whether ODN binding and cell uptake occurs through the same or a different mechanism from that of high molecular weight DNA.
Lymphocyte ODN uptake has been shown to be regulated by cell activation. Spleen cells stimulated with the B cell mitogen LPS had dramatically enhanced ODN uptake in the B cell population, while spleen cells treated with the T cell mitogen Con A showed enhanced ODN uptake by T but not B cells (Krieg, A. M., F. Gmelig-Meyling, M. F. Gourley, W. J. Kisch, L. A. Chrisey, and A. D. Steinberg. 1991. xe2x80x9cUptake of oligodeoxyribonucleotides by lymphoid cells is heterogeneous and inducible.xe2x80x9d Antisense Research and Development 1:161).
Several polynucleotides have been extensively evaluated as biological response modifiers. Perhaps the best example is poly (I,C) which is a potent inducer of IFN production as well as macrophage activator and inducer of NK activity (Talmadge, J. E., J. Adams, H. Phillips, M. Collins, B. Lenz, M. Schneider, E. Schlick, R. Ruffmann, R. H. Wiltrout, and M. A. Chirigos. 1985. xe2x80x9cImmunomodulatory effects in mice of polyinosinic-polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose.xe2x80x9d Cancer Res. 45:1058; Wiltrout, R. H., R. R. Salup, T. A. Twilley, and J. E. Talmadge. 1985. xe2x80x9cImmunomodulation of natural killer activity by polyribonucleotides.xe2x80x9d J. Biol. Respn. Mod. 4:512; Krown, S. E. 1986. xe2x80x9cInterferons and interferon inducers in cancer treatment.xe2x80x9d Sem. Oncol. 13:207; and Ewel, C. H., S. J. Urba, W. C. Kopp, J. W. Smith II, R. G. Steis, J. L. Rossio, D. L. Longo, M. J. Jones, W. G. Alvord, C. M. Pinsky, J. M. Beveridge, K. L. McNitt, and S. P. Creekmore. 1992. xe2x80x9cPolyinosinic-polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose in combination with interleukin-2 in patients with cancer: clinical and immunological effects.xe2x80x9d Canc. Res. 52:3005). It appears that this murine NK activation may be due solely to induction of IFN-p secretion (Ishikawa, R., and C. A. Biron. 1993. xe2x80x9cIFN inducation and associated changes in splenic leukocyte distributionxe2x80x9d. J. Immunol. 150:3713). This activation was specific for the robose sugar since deoxyribose was ineffective. Its potent in vitro antitumor activity led to several clinical trials using poly (I,C) complexed with poly-L-lysine and carboxymethylcellulose (to reduce degradation by RNAse) Talmadge, J. E., et al., 1985. cited supra; Wiltrout, R. H., et al, 1985. cited supra); Krown, S. E., 1986. cited supra); and Ewel, C. H., et al., 1992. cited supra). Unfortunately, toxic side effects have thus far prevented poly (I,C) from becoming a useful therapeutic agent.
Guanine ribonucleotides substituted at the C8 position with either a bromine or a thiol group are B cell mitogens and may replace xe2x80x9cB cell differentiation factorsxe2x80x9d (Feldbush, T. L., and Z. K., Ballas. 1985. xe2x80x9cLymphokine-like activity of 8-mercaptoguanosine: induction of T and B cell differentiation.xe2x80x9d J. Immunol. 134:3204; and Goodman, M. G. 1986. xe2x80x9cMechanism of synergy between T cell signals and C8-substituted guanine nucleosides in humoral immunity: B lymphotropic cytokines induce responsiveness to 8-mercaptoguanosine.xe2x80x9d J. Immunol. 136:3335). 8-mercaptoguanosine and 8-bromoguanosine also can substitute for the cytokine requirement for the generation of MHC restricted CTL (Feldbush, T. L., 1985. cited supra), augment murine NK activity (Koo, G. C., M. E. Jewell, C. L. Manyak, N. H. Sigal, and L. S. Wicker. 1988. xe2x80x9cActivation of murine natural killer cells and macrophages by 8-bromoguanosine.xe2x80x9d J. Immunol. 140:3249), and synergize with IL-2 in inducing murine LAK generation (Thompson, R. A., and Z. K. Ballas. 1990. xe2x80x9cLymphokine-activated killer (LAK) cells. V. 8-Mercaptoguanosine as an IL-2-sparing agent in LAK generation.xe2x80x9d J. Immunol. 145:3524). The NK and LAK augmenting activities of these C8-substituted guanosines appear to be due to their induction of IFN (Thompson, R. A., et al. 1990. cited supra0. Recently, a 5xe2x80x2 triphosphorylated thymidine produced by a mycobacterium was found to be mitogenic for a subset of human xcex3xcex4 T cells (Constant, P., F. Davodeau, M.-A. Peyrat, Y. Poquet, G. Puzo, M. Bonneville, and J.-J. Fournie. 1994. xe2x80x9cStimulation of human xcex3xcex4 T cells by nonpeptidic mycobacterial ligands.xe2x80x9d Science 264:267). This report indicated the possibility that the immune system may have evolved ways to preferentially respond to microbial nucleic acids.
Several observations suggest that certain DNA structures may also have the potential to activate lymphocytes. For example, Bell et al. reported that nucleosomal protein-DNA complexes (but not naked DNA) in spleen cell supernatants caused B cell proliferation and immunoglobulin secretion (Bell, D. A., B. Morrison, and P. VandenBygaart. 1990. xe2x80x9cImmunogenic DNA-related factors.xe2x80x9d J. Clin. Invest. 85:1487). In other cases, naked DNA has been reported to have immune effects. For example, Messina et al. have recently reported that 260 to 800 bp fragments of poly (dG).(dC) and poly (dG.dC) were mitogenic for B cells (Messina, J. P., G. S. Gilkeson, and D. S. Piesetsky. 1993. xe2x80x9cThe influence of DNA structure on the in vitro stimulation of murine lymphocytes by natural and synthetic polynucleotide antigens.xe2x80x9d Cell. Immunol. 147:148). Tokunaga, et al. have reported that dGdC induces xcex3-IFN and NK activity (Tokunaga, S. Yamamoto, and K. Nama. 1988. xe2x80x9cA synthetic single-stranded DNA, poly(dG, dC), induces interferon-xcex1/b and -g, augments natural killer activity, and suppresses tumor growth.xe2x80x9d Jpn. J. Cancer Res. 79:682). Aside from such artificial homopolymer sequences, Pisetsky et al. reported that pure mammalian DNA has no detectable immune effects, but that DNA from certain bacteria induces B cell activation and immunoglobulin secretion (Messina, J. P., G. S. Gilkeson, and D. S. Pisetsky. 1991. xe2x80x9cStimulation of in vitro murine lymphocyte proliferation by bacterial DNA.xe2x80x9d J. Immunol. 147:1759). Assuming that these data did not result from some unusual contaminant, these studies suggested that a particular structure or other characteristic of bacterial DNA renders it capable of triggering B cell activation. Investigations of mycobacterial DNA sequences have demonstrated that ODN which contain certain palindrome sequences can activate NK cells (Yamamoto, S., T. Yamamoto, T. Kataoka, E. Kuramoto, O. Yano, and T. Tokunaga. 1992. xe2x80x9cUnique palindromic sequences in synthetic oligonucleotides are required to induce INF and augment INF-mediated natural killer activity.xe2x80x9d J. Immunol. 148:4072Kuramoto, E., O. Yano, Y. Kimura, M. Baba, T.Makino, S. Yamamoto, T. Yamamoto, T. Kataoka, and T. Tokunaga. 1992. xe2x80x9cOligonucleotide sequences required for natural killer cell activation.xe2x80x9d Jpn. J. Cancer Res. 83:1128).
Several phosphorothioate modified ODN have been reported to induce in vitro or in vivo B cell stimulation (Tanaka, T., C. C. Chu, and W. E. Paul. 1992. xe2x80x9cAn antisense oligonucleotide complementary to a sequence in Ig2b increases g2b germline transcripts, stimulates B cell DNA synthesis, and inhibits immunoglobulin secretion.xe2x80x9d J. Exp. Med. 175:597; McIntyre, K. W., K. Lombard-Gillooly, J. R. Perez, C. Kunsch, U. M. Sarmiento, J. D. Larigan, K. T. Landreth, and R. Narayanan. 1993. xe2x80x9cA sense phosphorothioate oligonucleotide directed to the initiation codon of transcription factor NF-xcexaB T65 causes sequence-specific immune stimulation.xe2x80x9d Antisense Res. Develop. 3:309; and Pisetsky, D. S., and C. F. Reich. 1993. xe2x80x9cStimulation of murine lymphocyte proliferation by a phosphorothioate oligonucleotide with antisense activity for herpes simplex virus.xe2x80x9d Life Sciences 54:101). These reports do not suggest a common structural motif or sequence element in these ODN that might explain their effects.
The cAMP response element binding protein (CREB) and activating transcription factor (ATF) or CREB/ATF family of transcription factors is a ubiquitously expressed class of transcription factors of which 11 members have so far been cloned (reviewed on de Groot, R. P., and P. Sassone-Corsi: xe2x80x9cHormonal control of gene expression: Multiplicity and versatility of cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate-responsive nuclear regulators.xe2x80x9d Mol. Endocrin. 7:145, 1993; Lee, K. A. W., and N. Masson: xe2x80x9cTranscriptional regulation by CREB and its relatives.xe2x80x9d Biochim. Biophys. Acta 1174:221, 1993). They all belong to the basic region/leucine zipper (bZip) class of proteins. All cells appear to express one or more CREB/ATF proteins, but the members expressed and the regulation of mRNA splicing appear to be tissue-specific. Differential splicing of activation domains can determine whether a particular CREB/ATF protein will be a transcriptional inhibitor or activator. Many CREB/ATF proteins activate viral transcription, but some splicing variants which lack the activation domain are inhibitory. CREB/ATF proteins can bind DNA as homo- or hetero-dimers through the cAMP response element, the CRE, the consensus form of which is the unmethylated sequence TGACGTC (SEQ. ID. No. 103) (binding is abolished if the CpG is methylated) (Iguchi-Ariga, S. M. M., and W. Schaffner: xe2x80x9cCpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA (SEQ. ID. No.104) abolishes specific factor binding as well as transcriptional activation.xe2x80x9d Genese and Develop. 3:612, 1989.
The transcriptional activity of the CRE is increased during B cell activation (Xie. H., T. C. Chiles, and T. L. Rothstein: xe2x80x9cInduction of CREB activity via the surface Ig receptor of B cells.xe2x80x9d J. Immunol. 151:880, 1993). CREB/ATF proteins appear to regulate the expression of multiple genes through the CRE including immunologically important genes such as fos, jun B, Rb-1, IL-6, IL-1 (Tsukada, J., K. Saito, W. R. Waterman, A. C. Webb, and P. E. Auron: xe2x80x9cTranscription factors NF-IL6 and CREB recognize a common essential site in the human prointerleukin 1 gene.xe2x80x9d Mol. Cell. Biol. 14:7285, 1994; Gray, G. D., O. M. Hernandez, D. Hebel, M. Root, J. M. Pow-Sang, and E. Wickstrom: xe2x80x9cAntisense DNA inhibition of tumor growth induced by c-Ha-ras oncogene in nude mice.xe2x80x9d Cancer Res. 53:577, 1993), IFN- (Du, W., and T. Maniatis: xe2x80x9cAn ATF/CREB binding site protein is required for virus induction of the human interferon B gene.xe2x80x9d Proc. Natl. Acad. Sci. USA 89:2150, 1992), TGF-1 (Asiedu, C. K., L. Scott, R. K. Assoian, M. Ehrlich: xe2x80x9cBinding of AP-1/CREB proteins and of MDBP to contiguous sites downstream of the human TGF-B1 gene.xe2x80x9d Biochim. Biophys. Acta 1219:55, 1994), TGF-2, class II MHC (Cox, P. M., and C. R. Goding: xe2x80x9cAn ATF/CREB binding motif is required for aberrant constitutive expression of the MHC class II Dra promoter and activation by SV40 T-antigen.xe2x80x9d Nuc. Acids Res. 20:4881, 1992), E-selectin, GM-CSF, CD-8, the germline Ig constant region gene, the TCR V gene, and the proliferating cell nuclear antigen (Huang, D., P. M. Shipman-Appasamy, D. J. Orten, S. H. Hinrichs, and M. B. Prystowsky: xe2x80x9cPromoter activity of the proliferating-cell nuclear antigen gene is associated with inducible CRE-binding proteins in interleukin 2-stimulated T lymphocytes.xe2x80x9d Mol. Cell. Biol. 14:4233, 1994). In addition to activation through the cAMP pathway, CREB can also mediate transcriptional responses to changes in intracellular Ca++ concentration (Sheng, M., G. McFadden, and M. E. Greenberg: xe2x80x9cMembrane depolarization and calcium induce c-fos transcription via phosphorylation of transcription factor CREB.xe2x80x9d Neuron 4:571, 1990).
The role of protein-protein interactions in transcriptional activation by CREB/ATF proteins appears to be extremely important. There are several published studies reporting direct or indirect interactions between NFKB proteins and CREB/ATF proteins (Whitley, et al., (1994) Mol. and Cell. Biol. 14:6464; Cogswell, et al., (1994) J. Immun. 153:712; Hines, et al, (1993) Oncogene 8:3189; and Du, et al., (1993) Cell 74:887. Activation of CREB through the cyclic AMP pathway requires protein kinase A (PKA), which phosphorylates CREB341 on ser133 and allows it to bind to a recently cloned protein, CBP (Kwok, R. P. S., J. R. Lundblad, J. C. Chrivia, J. P. Richards, H. P. Bachinger, R. G. Brennan, S. G. E. Roberts, M. R. Green, and R. H. Goodman: xe2x80x9cNuclear protein CBP is a coactivator for the transcription factor CREB.xe2x80x9d Nature 370:223, 1994; Arias, J., A. S. Alberts, P. Brindle, F. X. Claret, T. Smea, M. Karin, J. Feramisco, and M. Montminy: xe2x80x9cActivation of cAMP and mitogen responsive genes relies on a common nuclear factor.xe2x80x9d Nature 370:226, 1994). CBP in turn interacts with the basal transcription factor TFIIB causing increased transcription. CREB also has been reported to interact with dTAFII 110, a TATA binding protein-associated factor whose binding may regulate transcription (Ferreri, K., G. Gill, and M. Montminy: xe2x80x9cThe cAMP-regulated transcription factor CREB interacts with a component of the TFIID complex.xe2x80x9d Proc. Natl. Acad. Sci. USA 91:1210, 1994). In addition to these interactions, CREB/ATF proteins can specifically bind multiple other nuclear factors (Hoeffler, J. P., J. W. Lustbadfer, and C.-Y. Chen: xe2x80x9cIdentification of multiple nuclear factors that interact with cyclic adenosine 3xe2x80x2,5xe2x80x2-monophosphate response element-binding protein and activating transcription factor-2 by protein-protein interactions.xe2x80x9d Mol. Endocrinol. 5:256, 1991) but the biologic significance of most of these interactions is unknown. CREB is normally thought to bind DNA either as a homodimer or as a heterodimer with several other proteins. Surprisingly, CREB monomers constitutively activate transcription (Krajewski, W., and K. A. W. Lee: xe2x80x9cA monomeric derivative of the cellular transcription factor CREB functions as a constitutive activator.xe2x80x9d Mol. Cell. Biol. 14:7204, 1994).
Aside from their critical role in regulating cellular transcription, it has recently been shown that CREB/ATF proteins are subverted by some infectious viruses and retroviruses, which require them for viral replication. For example, the cytomegalovirus immediate early promoter, one of the strongest known mammalian promoters, contains eleven copies of the CRE which are essential for promoter function (Chang, Y.-N., S. Crawford, J. Stall, D. R. Rawlins, K.-T. Jeang, and G. S. Hayward: xe2x80x9cThe palindromic series I repeats in the simian cytomegalovirus major immediate-early promoter behave as both strong basal enhancers and cyclic AMP response elements.xe2x80x9d J. Virol. 64:264, 1990). At least some of the transcriptional activating effects of the adenovirus E1A protein, which induces many promoters, are due to its binding to the DNA binding domain of the CREB/ATF protein, ATF-2, which mediates E1A inducible transcription activation (Liu, F., and M. R. Green: xe2x80x9cPromoter targeting by adenovirus E1A through interaction with different cellular DNA-binding domains.xe2x80x9d Nature 368:520, 1994). It has also been suggested that E1A binds to the CREB-binding protein, CBP (Arany, Z., W. R. Sellers, D. M. Livingston, and R. Eckner: xe2x80x9cE1A-associated p300 and CREB-associated CBP belong to a conserved family of coactivators.xe2x80x9d Cell 77:799, 1994). Human T lymphotropic virus-I (HTLV-1), the retrovirus which causes human T cell leukemia and tropical spastic paresis, also requires CREB/ATF proteins for replication. In this case, the retrovirus produces a protein, Tax, which binds to CREB/ATF proteins and redirects them from their normal cellular binding sites to different DNA sequences (flanked by G- and G-rich sequences) present within the HTLV transcriptional enhancer (Paca-Uccaralertkun, S., L.-J. Zhao, N. Adya, J. V. Cross, B. R. Cullen, I. M. Boros, and C.-Z. Giam: xe2x80x9cIn vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator, Tax.xe2x80x9d Mol. Cell. Biol. 14:456, 1994; Adya, N., L.-J. Zhao, W. Huang, I. Boros, and C.-Z. Giam: xe2x80x9cExpansion of CREB""s DNA recognition specificity by Tax results from interaction with Ala-Ala-Arg at positions 282-284 near the conserved DNA-binding domain of CREB.xe2x80x9d Proc. Natl. Acad. Sci. USA 91:5642, 1994).
The present invention is based on the finding that certain nucleic acids containing unmethylated cytosine-guanine (CpG) dinucleotides activate lymphocytes in a subject and redirect a subject""s immune response from a Th2 to a Th1 (e.g., by inducing monocytic cells and other cells to produce Th1 cytokines, including IL-12, IFN-xcex3 and GM-CSF). Based on this finding, the invention features, in one aspect, novel immunostimulatory nucleic acid compositions.
In one embodiment, the invention provides an isolated immunostimulatory nucleic acid sequence containing a CpG motif represented by the formula:
5xe2x80x2N1X1CGX2N23xe2x80x2
wherein at least one nucleotide separates consecutive CpGs; X1 is adenine, guanine, or thymine; X2 is cytosine or thymine; N is any nucleotide and N1+N2 is from about 0-26 bases with the proviso that N1 and N2 do not contain a CCGG quadmer or more than one CCG or CGG trimer; and the nucleic acid sequence is from about 8-30 bases in length.
In another embodiment, the invention provides an isolated immunostimulatory nucleic acid sequence contains a CpG motif represented by the formula:
5xe2x80x2N1X1X2CGX3X4N2Cxe2x80x2
wherein at least one nucleotide separates consecutive CpGs; X1X2 is selected from the group consisting of GpT, GpG, GpA, ApT and ApA; X3 X4 is selected from the group consisting of TpT or CpT; N is any nucleotide and N1+N2 is from about 0-26 bases with the proviso that N1 and N2 do not contain a CCGG quadmer or more than one CCG or CGG trimer; and the nucleic acid sequence is from about 8-30 bases in length.
In another embodiment, the invention provides a method of stimulating immune activation by administering the nucleic acid sequences of the invention to a subject, preferably a human. In a preferred embodiment, the immune activation effects predominantly a Th1 pattern of immune activation.
In another embodiment, the nucleic acid sequences of the invention stimulate cytokine production. In particular, cytokines such as IL-6, IL-12, IFN-xcex3, TNF-xcex1 and GM-CSF are produced via stimulation of the immune system using the nucleic acid sequences described herein. In another aspect, the nucleic acid sequences of the invention stimulate the lytic activity of natural killer cells (NK) and the proliferation of B cells.
In another embodiment, the nucleic acid sequences of the invention are useful as an artificial adjuvant for use during antibody generation in a mammal such as a mouse or a human.
In another embodiment, autoimmune disorders are treated by inhibiting a subject""s response to CpG mediated leukocyte activation. The invention provides administration of inhibitors of endosomal acidification such as bafilomycin a, chloroquine, and monensin to ameliorate autoimmune disorders. In particular, systemic lupus erythematosus is treated in this manner.
The nucleic acid sequences of the invention can also be used to treat, prevent or ameliorate other disorders (e.g., a tumor or cancer or a viral, fungal, bacterial or parasitic infection). In addition, the nucleic acid sequences can be administered to stimulate a subject""s response to a vaccine. Furthermore, by redirecting a subject""s immune response from Th2 to Th1, the claimed nucleic acid sequences can be used to treat or prevent an asthmatic disorder. In addition, the claimed nucleic acid molecules can be administered to a subject in conjunction with a particular allergen as a type of desensitization therapy to treat or prevent the occurrence of an allergic reaction associated with an asthmatic disorder.
Further, the ability of the nucleic acid sequences of the invention described herein to induce leukemic cells to enter the cell cycle supports their use in treating leukemia by increasing the sensitivity of chronic leukemia cells followed by conventional ablative chemotherapy, or by combining the nucleic acid sequences with other immunotherapies.
Other features and advantages of the invention will become more apparent from the following detailed description and claims.